Preparation of Joint Extracts.

Since mice are widely used to establish rheumatoid arthritis models, assessment of the pathogenesis of local arthritis is fundamental. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function. The first step in pathogenesis-related protein analysis is joint tissue extraction. Unlike other large rodents, obtaining synovium from model mice is challenging since it is so small and fragile. In this chapter, methods for harvesting synovium through a quadriceps approach and preparing protein extracts are introduced.

Denervation-Induced Sarcopenia Model.

Rheumatoid arthritis (RA) is an important risk factor for sarcopenia. Physical inactivity, systemic inflammatory factors, and medication directly or indirectly induce skeletal muscle loss in RA patients. The sarcopenia-induced systemic or local proinflammatory microenvironment also contributes to the onset and progression of autoimmune disease. Accumulated evidence suggests the importance of treatment and management of sarcopenia in patients with RA to improve their long-term prognosis. To elucidate the relationship between skeletal muscle and systemic immune homeostasis, a denervation-induced skeletal muscle-losing mouse model is introduced in this chapter. By developing local amyotrophy in the sciatic nerve-dominant area in a RA model, the underlying mechanism of sarcopenia in RA could be assessed. Also, an examination of the efficacy of anti-rheumatic regents on sarcopenia and the influence of sarcopenia management on RA improvement is also achievable.

Production of Neutralizing Antibody.

Techniques employing monoclonal antibodies (mAbs) are widely used in the initial development phase of biologics. The usefulness of mAbs in basic RA research has been established based on their characteristics, including specificity of binding, homogeneity, and ability to be produced on a large scale. MAb immunoglobulins are the starting material for the generation of smaller antibody fragments and other engineered immunomodulatory antibodies. In this chapter, the basic hybridoma technique, which is a well-established and feasible method for the production of mAbs involving animal immunization, cell fusion, hybridoma screening, expanding positive hybridomas, and purification, is introduced. Aiming at specific affinity to a membrane protein, synthetic proteoliposomes are used in the immunization and screening steps.

Screening of Ca2+ Influx in Lymphocytes.

The Ca2+ ion is an important second messenger in lymphocytes, similarly to its function in other mammalian cells. The generation of long-lasting intracellular Ca2+ elevations is essential for Ca2+-dependent gene transcription, proliferation, differentiation, and cytokine production in lymphocytes. Since store-operated Ca2+ entry (SOCE) is considered the predominant mode of Ca2+ influx in lymphocytes, the activation and function of lymphocytes can be generally predicted by monitoring SOCE. A method suitable for dynamic monitoring of Ca2+ influx using fura-2 labeling in lymphocytes is introduced in this chapter. Using this technique, large-scale screening of the activation status of primary or cultured lymphocytes can be realized.

Dietary leucine supplementation restores T-cell mitochondrial respiration and regulates T-lineage differentiation in denervation-induced sarcopenic mice.

With the aim of offsetting immune dysfunction preceded by sarcopenia, the feasibility and efficiency of nutritional leucine supplementation were evaluated using a murine denervation-induced sarcopenia model. Sciatic nerve axotomy caused significant loss of skeletal muscle of the hind limbs and accelerated mitochondrial stress along with suppressed ATP production in spleen-derived T cells. Dietary leucine intake not only ameliorated muscle mass anabolism in a sarcopenic state, but also restored mitochondrial respiratory function, as indicated by elevated levels of basal respiration, maximal respiration, spare respiratory capacity, and ATP production, in T cells, which in turn led to downregulated expression of mTOR and downstream signals, as indicated by the findings of comprehensive transcriptome analysis. Consequentially, this finally resulted in amelioration of the sarcopenia-induced relative Th1/Th17-dominant proinflammatory microenvironment. These results highlight the importance of leucine-promoted metabolic cues in directing T cell fate in a sarcopenic microenvironment. The present study provides insights that particularly help rationalize the design and optimization of leucine supplementation for chronic sarcopenic patients with autoimmune diseases.

Exploring an immortal Turritopsis sp. as a less conventional natural system for study of aging.

Aim: Given its excellent capability of escaping from unavoidable harm and death, Turritopsissp. (T. sp.) has captured the attention and fascination of scientists as a less conventional tool for aging research. The current study introduces a method for establishment of a research model and comprehensive transcriptomic analysis to reveal the structural and functional diversity of T. sp. Methods: T. sp. medusae collected from the Pacific Ocean near Japan were reared using a common laboratory setting. Tissues of the gastrovascular cavity part (GP) and nerve ring part (NP) were collected, and total RNA was extracted. Bulk RNA-seq was performed to compare the different transcriptome landscapes between GP and NP. Results: The GP fragment could be utilized for studies related to stress response and systemic senescence, while the NP fragment could be used to explore system rejuvenation, self-repair and regeneration. Conclusions: As a less conventional system for aging research, by employing the most recently developed tools and techniques in the genomic revolution, comprehensive elucidation of the composition, development, and functions of T. sp. enabled us to explore the underlying mechanisms of the response to environmental stress and rejuvenation.

Intermittent environmental exposure to hydrogen prevents skin photoaging through reduction of oxidative stress.

AIM: Molecular hydrogen is not only expected to be used as an energy-generating resource, but also to have preventive effects on a variety of clinical manifestations related to oxidative stress through scavenging radicals or regulating gene expression. In the current study, we investigated the influence of intermittent environmental exposure to hydrogen gas at a safe concentration (1.3%) on photoaging using an ultraviolet A (UVA)-irradiated murine model. METHODS: To mimic the expected human daily activity cycle, UVA exposure in the daytime and hydrogen exposure in the night-time, an original design, UVA-transmission, hydrogen-exposure system was established. Mice were bred under experimental conditions of UVA irradiation and normal air for 8 h (outdoor time 09.00-17.00 hours), and UVA non-irradiation and inhalation of hydrogen gas for 16 h (indoor time 17.00-09.00 hours), and the daily cycle was continued for up to 6 weeks. The progression of photoaging, including morphological changes, collagen degradation and UVA-related DNA damage, was evaluated. RESULTS: Intermittent administration of hydrogen gas by our system prevented UVA-induced epidermal signs, such as hyperplasia, melanogenesis and appearance of senescence cells, and UVA-induced dermal signs, such as collagen degradation. In addition, we detected attenuation of DNA damage in the hydrogen exposure group as indirect evidence that intermittent exposure to hydrogen gas reduced oxidative stress. CONCLUSIONS: Our findings support the notion that long-term, intermittent environmental exposure to hydrogen gas in daily life has a beneficial effect on UVA-induced photoaging. Geriatr Gerontol Int 2023; 23: 304-312.

Generation of DNA-aptamers targeting galectin-7 for the identification of cholesteatoma residue.

PURPOSE: Aiming at complete excision of cholesteatoma during trympanomastoidectomy and therefore reducing the risk of recurrence, intraoperative imaging techniques are required to assist the visualization of cholesteatoma residue. Galectin-7 has been demonstrated to be a biomarker for cholesteatoma matrix and used for intraoperatively identifying the excision margins. METHODS: A galectin-7-targeted DNA-aptamer library was generated for labeling the cholesteatoma matrix using cell-systematic evolution of ligands by an exponential enrichment technique. The binding characteristics of the identified aptamers were analyzed, and structure optimization of the identified aptamers was carried out both in silico and in vitro. FINDINGS: A fluorophore-labeled structure-optimized DNA fragment was commercially synthesized as a non-invasive aptamer-based probe for intraoperative lesion detection. Using galectin-7-aptamer-guided molecular imaging, the excision margins of cholesteatoma matrix and surrounding normal tissue were successfully achieved within 15-20 min. CONCLUSIONS: Galectin-7-targeted aptamers could benefit molecular imaging-guided surgical treatment, which would enable clinicians to not only intraoperatively detect the locations of cholesteatoma matrix in the middle ear, but also assess the postoperative response of the expression profile to therapy. It is highly expected that further efforts for rational design and development should be directed towards the development of clinically translatable aptamer-based imaging agents.

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex.

The pulmonary pathological findings associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result from the release of multiple proinflammatory cytokines, which causes the subsequential damage of the lungs. The current study was undertaken to investigate the responses of mast cells to viral inoculation and their contribution to host defenses from the point of view of viral entry. Pseudovirions, in which the spike glycoprotein of SARS-CoV-2 was incorporated, triggered activation of mast cells, and a mast cell-derived chymase, MCP2, formed a complex with spike protein, which promoted protease-dependent viral entry. According to the quantification results of viral entry, 10 µM quercetin, a mast cell stabilizer, potentially potently inhibited 41.3% of viral entry, while 100 µM chymostatin, which served as a chymase inhibitor, suppressed 52.1% of viral entry, compared to non-treated cells. Study using mast cell-deficient mice showed that the absence of mast cells may influence early viral loading in the upper respiratory tract, which consequently increases the risk of viral invasion into the lower respiratory system. Furthermore, mast cell-deficient mice exhibited ongoing infection in the late phase post-viral inoculation, while clearance of virus-positive cells was observed in wild-type mice. In conclusion, mast cells act as a multifaceted immune modulator that is equipped with both protective effects and pathogenic influences on viral entry of SARS-CoV-2. The utility of mast cell stabilizers and chymase inhibitors in the treatment of SARS-CoV-2-induced acute respiratory syndrome should be optimized regarding the infection stage and the risk of cytokine storm.

Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress.

Molecular hydrogen is thought to have an inhibitory effect on oxidative stress, thereby attenuating the onset and progression of various diseases including cardiovascular disease; however, few reports have assessed the preventive effect of constitutive inhalation of hydrogen gas on of vascular remodeling. Here, we investigated the effect of constitutive inhalation of hydrogen gas on vascular neointima formation using a cuff-induced vascular injury mouse model. After constitutive inhalation of compressed hydrogen gas (O2 21%, N2 77.7%, hydrogen 1.3%) or compressed air only (O2 21%, N2 79%) by C57BL/6 mice for 2 weeks from 8 weeks of age in a closed chamber, inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia, and hydrogen gas administration was continued until sampling of the femoral artery. Neointima formation, accompanied by an increase in cell proliferation, was significantly attenuated in the hydrogen group compared with the control group. NADPH oxidase NOX1 downregulation in response to cuff injury was shown in the hydrogen group, but the expression levels of NADPH oxidase subunits, p40phox and p47phox, did not differ significantly between the hydrogen and control groups. Although the increase in superoxide anion production did not significantly differ between the hydrogen and control groups, DNA damage was decreased as a result of reduction of reactive oxygen species such as hydroxyl radical (⋅OH) and peroxynitrite (ONOO-) in the hydrogen group. These results demonstrate that constitutive inhalation of hydrogen gas attenuates vascular remodeling partly via reduction of oxidative stress, suggesting that constitutive inhalation of hydrogen gas at a safe concentration in the living environment could be an effective strategy for prevention of vascular diseases such as atherosclerosis.

Preparation of Joint Extracts.

Since mice are widely used to establish rheumatoid arthritis models, assessment of the pathogenesis of local arthritis is fundamental. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function. The first step in pathogenesis-related protein analysis is joint tissue extraction. Unlike other large rodents, obtaining synovium from model mice is challenging, since it is so small and fragile. In this chapter, methods for harvesting synovium through a quadriceps approach and preparing protein extracts are introduced.

Production of Neutralizing Antibody.

Techniques employing monoclonal antibodies (mAbs) are widely used in the initial development phase of biologics. The usefulness of mAbs in basic RA research has been established based on their characteristics, including specificity of binding, homogeneity, and ability to be produced on a large scale. MAb immunoglobulins are the starting material for the generation of smaller antibody fragments and other engineered immunomodulatory antibodies. In this chapter, the basic hybridoma technique, which is a well-established and feasible method for the production of mAbs involving animal immunization, cell fusion, hybridoma screening, expanding positive hybridomas, and purification, is introduced. Aiming at specific affinity to a membrane protein, synthetic proteoliposomes are used in the immunization and screening steps.

Screening of Ca2+ Influx in Lymphocytes.

The Ca2+ ion is an important second messenger in lymphocytes, similarly to its function in other mammalian cells. The generation of long-lasting intracellular Ca2+ elevations is essential for Ca2+-dependent gene transcription, proliferation, differentiation, and cytokine production in lymphocytes. Since store-operated Ca2+ entry (SOCE) is considered the predominant mode of Ca2+ influx in lymphocytes, the activation and function of lymphocytes can be generally predicted by monitoring SOCE. A method suitable for dynamic monitoring of Ca2+ influx using fura-2 labeling in lymphocytes is introduced in this chapter. Using this technique, large-scale screening of the activation status of primary or cultured lymphocytes can be realized.

Zoledronate modulates intracellular vesicle trafficking in mast cells via disturbing the interaction of myosinVa/Rab3a and sytaxin4/VAMP7.

Nitrogen-containing bisphosphonates (NBPs) have been widely used as bone anti-resorptive drugs for the treatment of osteoclast-dependent bone disorders. Zoledronate is currently the most potent NBP, and has potential as an inhibitor of farnesyl pyrophosphate synthase. The present study was undertaken to elucidate the possible effects of zoledronate on FcεRI-dependent mast cell activity in vitro, which is essential for in maintaining homeostasis of the gastrointestinal mucosa. Treatment with zoledronate significantly diminished exocytosis of mast cells, which was reflected by a decrease of FcεRI-dependent histamine release compared to that in vehicle-treated mast cells. Our single-vesicle monitoring and biochemical results suggested that zoledronate modulates intracellular formation of the myosinVa/Rab3a complex and syntaxin4/VAMP7 complex, which are critical in vesicle motility, and therefore disturbs exocytosis via suppression of the velocity of intracellular vesicles and inhibition of membrane fusion. Our findings imply that oral administration of zoledronate could modulate mucosal immune function by blocking mast cell function, and this risk should be of concern in the clinical usage of NBPs.

Efficacy of constant long-term delivery of YM-58483 for the treatment of rheumatoid arthritis.

The aim of this study was to investigate the efficacy and safety of YM-58483, a small molecular antagonist of Ca2+ release-activated Ca2+ (CRAC) channels, for the treatment of rheumatoid arthritis (RA), in vivo and ex vivo. YM-58483 was continuously injected subcutaneously in a collagen-induced arthritis (CIA) mouS.E.M.odel using an implanted osmotic pump. The severity of CIA was evaluated using the following parameters: body weight, hind paw volume, clinical score, histological analysis, cytokine levels, Ca2+ influx, and specific IgG production. The efficacy of long-term application of YM-58483 was also verified ex vivo in RA patient-derived peripheral blood monocytes. Assessment of the clinical severity of CIA, cytokine profile in serum and joint protein extracts, and specific IgG production showed that continuous application of YM-58483 suppressed synovial inflammation by inhibiting immune cell activity. Chemical screening and hepatography indicated that long-term subcutaneous delivery of YM-58483 was safer than oral administration for systemic application. Moreover, constant preincubation with YM-58483 at an IC50 of 0.1-1 nM altered proinflammatory cytokine production ex vivo in peripheral T cells derived from RA patients. Our findings suggest that continuous long-term application of appropriate CRAC inhibitors such as YM-58483 is a potential therapeutic strategy for global immunosuppression in RA.

Efficiency and Safety of CRAC Inhibitors in Human Rheumatoid Arthritis Xenograft Models.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483. Systemically constant, s.c. infused CRAC inhibitors showed anti-inflammatory activity in a human-NOD/SCID xenograft RA model as well as protective effects against the destruction of cartilage and bone. hCRACM1-IgG appeared to be safe for systemic application, whereas YM-58483 showed hepatic and renal toxicity in xenograft mice. Treatment with both CRAC inhibitors also caused hyperglycemia in xenograft mice. These results indicate the potential of hCRACM1-IgG and YM-58483 as anti-immunological agents for the treatment of RA. However, some safety issues should be addressed and application methods should be optimized prior to their clinical use.

Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis.

Abnormal store-operated calcium uptake has been observed in peripheral T lymphocytes of rheumatoid arthritis (RA) patients, and sustained intracellular calcium signalling is known to mediate the functions of many types of immune cells. Thus, it is hypothesized that regulating calcium entry through CRACM1 (the pore-forming subunit of calcium release-activated calcium (CRAC) channels; also known as ORAI1) may be beneficial for the management of RA. Localized CRACM1 knockdown in the joints and draining lymph nodes (DLNs) of mice with collagen-induced arthritis (CIA) was achieved via lentiviral-based delivery of shRNA targeting mouse CRACM1. Consistent with CRACM1 knockdown, calcium influx in synovial cells and the histopathological features of CIA were reduced. These effects were also associated with reduced levels of several notable inflammatory cytokines, such as IL-6, IL-17A, and IFN-γ, in the joints. Additionally, CRACM1-shRNA reduced the number of bone marrow-derived osteoclasts in vitro as well as osteoclasts in CIA joints, which was associated with reduced RANKL levels in the serum and joints. In summary, inhibiting calcium entry by CRACM1 knockdown suppressed arthritis development and may be therapeutically beneficial for RA patients.

CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca(2+)-independent manner via molecular interaction with syntaxin4.

Ca(2+) release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca(2+)-selective channels. In a non-excitable exocytosis model, we found that the extracellular L3 domain and the cytoplasmic C-terminus of CRACM3 interacted in an activity-dependent manner with the N-peptide of syntaxin4, a soluble N-ethylmaleimide-sensitive factor attachment receptor protein. Our biochemical, electrophysiological and single-vesicle studies showed that knockdown of CRACM3 suppressed functional exocytosis by decreasing the open time of the vesicle fusion pore without affecting Ca(2+) influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca(2+), led to an impaired Cm change, decreased the number of fusion events, and prolonged the dwell time of the fusion pore. CRACM3 changes the stability of the vesicle fusion pore in a manner consistent with the altered molecular expression. Our findings imply that CRACM3 plays a greater role in exocytosis than simply acting as a compensatory subunit of a Ca(2+) channel.

Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collagen-induced arthritis.

Immune cells, including T cells, B cells, and osteoclasts, in conjunction with their associated cytokines, have been studied as primary molecular therapeutic targets for the management of rheumatoid arthritis (RA) patients. The increase in cytosolic Ca(2+) levels through the activation of store-operated Ca(2+) release-activated channels (CRACs) is involved in mediating a disparate array of cellular responses by these immune cells. This study was undertaken to investigate the feasibility and efficiency of the regulation of Ca(2+) entry in the treatment of RA. To moderately suppress Ca(2+) entry via CRACs, we gene silenced CRACM3, which was induced by systemic application of specific short hairpin RNAs (shRNAs) using a lentiviral-delivery system, in a murine model of collagen-induced arthritis (CIA). The inflammatory responses were determined by measuring the levels of a panel of cytokines and chemokines in the joints and serum. Ag-specific responses were evaluated by determining the cytokine profile of T cells stimulated with autoantigen. We also analyzed the ability of specific CRACM3-shRNA to regulate mature osteoclast function in CIA mice. The therapeutic effect of lentiviral-delivered CRACM3-shRNA was associated with gene silencing of CRACM3, along with the successful biodistribution of the virus. Extracellular Ca(2+) influx in the splenocytes, thymocytes, and knee joint synovial cells was moderately suppressed. Inflammatory responses and autoimmune responses were reduced by CRACM3 gene silencing. A decrease in mature osteoclast activity also was observed in CRACM3-shRNA-treated CIA mice. These results indicate that regulation of Ca(2+) entry through lentivirus-mediated CRACM3 gene silencing is beneficial in the treatment of RA.